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Western Blot by Gaelin Kopec-Belliveau
Western Blot The western blot is a method used to separate and identify proteins using a polyacrylamide gel, electric currents and antibody detection. To begin the process, samples containing the protein of interest are broken down, usually using a blender or another device. Once the sample has been pureed, additional salts, buffers or detergents are added to the mixture to increase the rate of lysis of cells. The idea is to remove the secondary and tertiary structures of the proteins so they can be separated by their molecular weight. In many cases, protease and phosphatase inhibitors are added to ensure against the destruction of the sample by its own enzymes. This process is best done at a cold temperature to further prevent the degradation of the protein mixture. The next step in western blotting is gel electrophoresis. In this process, the denatured protein mixture is treated with buffers containing sodium dodecyl sulfate (SDS) that cling to the proteins and give them a negative charge. They are then placed on one side of a sample of polyacrylamide gel and pulled through the pores in the gel by a positively charged electrode on the opposite side. Smaller molecules will be pulled faster and will move through smaller pores than larger molecules. The proteins are loaded into the gel in small wells at the end opposite to the electrode. One well is filled with markers or ladders which are commercially prepared mixtures of proteins with known molecular weights and stained for easy visualization. This is used to compare the sample proteins to, in order to aid in identification. When the voltage is applied at the electrode end of the gel, the molecule deposits created from differing rates of advancement of differently sized molecules are called bands. Once the proteins have been separated in the gel, they are usually transferred onto a membrane through a process called electroblotting. This involves pulling the protein onto the membrane using an electric current. Once the proteins have adhered to the membrane it is treated with a dilute solution containing animal derived proteins that attach to the membrane in all the places where the target protein is not attached. This ensures that when the antibody is added it will not attach to the membrane but to the target protein. The process of tagging the proteins involves two steps. First the membrane is incubated in a solution containing antibodies that will directly bind to the target protein for anywhere from 30 minutes to overnight. After the incubation, the membrane is removed and any unbound antibodies are removed. Next the membrane is washed with another antibody meant to bind to the already bound antibodies on the target proteins. There are several ways of detecting these attached antibodies in order to identify the proteins of interest but the most common way is using a chemiluminescent agent that, when reacted, will produce luminescence proportionate to the amount of protein present. By exposing the membrane to light, it is possible to determine what types and amounts of protein were present in the sample. 4 History and Applications The western blot was a technique first proposed by Harry Towbin, a scientist working at the Friedrich Miescher Institute in the 1970's. Towbin was working with ribosomes and having problems with protein analysis. The trouble was there was no way to distinguish specific proteins. Towbin's solution allowed the separation of proteins by size and various other characteristics. Completely revolutionizing protein separation in the scientific community. 1 The western blot is a technique with many real world applications. First, the process can determine the nature of the epitope or protein in question. It is also extremely useful in epitope mapping. Epitope mapping identifies the process of binding sites of antibodies on their target antigens. This can aid in the the development of therapeutics and vaccines. Additionally, the western blot can be used to learn more about amino acid composition and sequence analysis. The western blot is extremely prevalent in the world of science today. The process is used as a confirmatory HIV-test. Known HIV-infected cells are blotted and are incubated with human serum. If there are anti-HIV antibodies in the human serum they will attach to the HIV-infected cells, providing a diagnosis. Additionally, western blotting is used to detect HBV. The length of the DNA molecules coding for the hepatitis B virus is known to be roughly 3.2 kbp long. By using the western blot technique it is possible to detect these pieces of DNA and successfully diagnose the disease. 3 Original Literature Below is a link to the origianl literature describing the western blot method. It was publishdd by Harry Towbin, Theophil Staehelin and Julian Gordon in 1979. http://www.pnas.org/content/76/9/4350.full.pdf References 1 Mukhopadhyay, R. (2012). The Men Behind Western Blotting 2014, from http://www.asbmb.org/asbmbtoday/asbmbtoday_article.aspx?id=16084&page_id=1 2 Structural Biochemistry/Proteins/Western Blotting. (2014), from http://en.wikibooks.org/wiki/Structural_Biochemistry/Proteins/Western_Blotting 3 Western Blot Applications for Medical Diagnosis (2004). ''Western Blot Encyclopeddia ''2014, from http://www.western-blot.us/applications-of-western-blotting/applications-in-medical-diagnosis 4 http://en.wikipedia.org/wiki/Western_blot